Pharmaceutical compositions for the treatment of diseases related to neurotrophines

ABSTRACT

The present invention refers to pharmaceutical preparations including as active compounds 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I) and/or their dimers of general formula (II) and (III) acting as agonists of human neurotrophines. Therefore, such compounds of formula (I), (II) and (III) are useful for treatment of diseases in which the neurotrophine functions are involved in defect, particularly of Nerve Growth Factor (NGF), such as neurodegenerative diseases of central nervous system (CNS), acquired immunodeficiency due to a reduced NGF biodisponibility, or morbous conditions in which the stimulus of neoangiogenesis process is convenient.

This application is a U.S. national stage application under 35 U.S.C. §371 of PCT/EP2003/006471, filed Jun. 18, 2003, which claims priority from Italian application number FI2002A000107, filed Jun. 19, 2002.

FIELD OF THE INVENTION

The present invention refers to pharmaceutical compositions comprising 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I), their dimers of general formula (II) or (III) hereinafter reported, or mixtures thereof, useful in the treatment of pathologies in which the neurotrophine functions, particularly of Nerve Growth Factor (NGF), are altered.

STATE OF THE ART

Numerous proteins and polypeptidic factors regulate cell growth and/or survival. The first of such factors which was identified and functionally characterised is NGF. Later on, other proteins belonging to the same NGF family were identified that exert their activity on different populations of nervous cells. All these proteins is are collectively referred to as “neurotrophins”.

NGF, upon interaction with specific surface receptors, prevents neuronal cell death during embryonal development and throughout adult life. NGF administration was proven advantageous in pathological conditions, such as degenerative and ischaemic disorders of Central Nervous System (CNS), spinal lesions, and toxicity of excitory amino acids. In fact, together with other neurotrophic factors, NGF promotes neuronal regeneration and supports neuronal functions.

Therapeutic uses of NGF have been limited by its poor ability to get across the blood-brain-barrier, partly due to the molecular size of the native factor. Thus, the development of non-peptidic compounds able to specifically mimic the activities of the natural ligand is a useful approach to obviate such limitations. Relevant examples of such compounds are a) phorbol esters, that mimic NGF presumably by modifying PKCc activity; b) ganglioside and other unrelated lipidic compounds, that promote neuritic outgrowth from dorsal root ganglia, or other sympathetic, neurones; c) Triap (1,1,3-triciano-2-ammino-1-propene), a small compound able to support survival and induce neuritic growth in PC12 cells. In all of the above cases, activity of molecules is not mediated by interactions with NGF receptors. Development of new non-peptidic compounds able to interact with specific receptors, thus behaving as agonists or antagonists, of human neurotrophins is of utmost importance, since they may be used as drugs for treatment of disorders related to a defective or excessive activity of neurotrophins.

SUMMARY OF THE INVENTION

Now, the Applicants have unexpectedly found that 3-aza-bicyclo[3.2.1.]octane derivatives of general formula (I) and their dimers of general formula (II) and (III) as reported hereinafter, are active as agonists of human neurotrophines, therefore they are useful for preparation of pharmaceutical compositions for the treatment of diseases in which the neurotrophine functions, particularly the NGF functions, are involved in defect.

It is therefore subject of the present invention a pharmaceutical composition comprising as the active principle at least one among the 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I), or their dimers of general formula (II) and (III), or mixtures thereof:

wherein: R₁ and R′₁, equal or different between each other, are selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, heterocycle, arylC₁₋₈alkyl; heterocycleC₁₋₈alkyl, RR′N—C₁₋₈alkyl, RR′N-aryl, FmocNR′-aryl, BocNR′-aryl, CBzNR′-aryl, RO-aryl, R(O)C-aryl, RO(O)C-aryl, RR′N(O)C-aryl; FmocNR′—C₁₋₈alkyl, BocNR′—C₁₋₈alkyl, CbzNR′—C₁₋₈alkyl, FmocNR′—C₁₋₈aryl, BocNR′—C₁₋₈aryl and CbzNR′—C₁ aryl, R₂ and R′₂, equal or different between each other, are selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, arylC₁₋₈alkyl, heterocycleC₁₋₈alkyl, aminoC₁₋₈alkyl, aminoaryl, C₁₋₈alkyloxyaryl, hydroxyaryl, hydroxyC₁₋₈alkyl, carboxyC₁₋₈alkyl, methyloxycarbonylC₁₋₈alkyl, carboxyaryl, carboalkyloxyaryl, alkylcarbamoylaryl and -(side chains of amino acids), or R₁ and R₂, taken together, and R₁′ and R₂′, taken together, are C₁₋₄alkyl, C₂₋₄ alkenyl, cycloalkyl or benzofused cycloalkyl, to form a bridge of 3, 4, 5, 6 terms, R₃ and R₃′ are selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, arylC₁₋₈alkyl, heterocycleC₁₋₈alkyl, RR′NC₁₋₈alkyl, RR′Naryl, RO—C₁₋₈alkyl, RO(O)C—C₁₋₈alkyl, R(O)C—C₁₋₈alkyl, RC(O)O—C₁₋₈alkyl, RC(O)N(R)C₁₋₈alkyl, RO-aryl, RO(O)C-aryl, R(O)C-aryl RC(O)O-aryl, RC(O)N(R)aryl, —CH(amino acid side-chain)CO₂R, —CH(amino acid side-chain)C(O)NR, —CH(CO₂R)— amino acid side-chain, CH(CONRR′)— amino acid side-chain, Fmoc, Boc and Cbz, R₄, R′₄ R₅, and R′₅, equal or different amongst each other, are selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alchenyl, C₂₋₈alchinyl, cycloalkyl, aryl, heterocycle, arylC₁₋₈alkyl and heterocycleC₁₋₈alkyl, R₆ is selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, arylC₁₋₈alkyl, heterocycle, heterocycleC₁₋₈alkyl; —C(O)R, —C(O)OR, —C(O)NRR′, CH₂OR, CH₂NRR′, —C(O)NH—CH(amino acid side-chain)C(O)OR, CH₂NR-Fmoc, CH₂NR-Boc and CH₂NR—CBz, R and R′, equal or different between each other, are selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, heterocycle, arylC₁₋₈alkyl; heterocycleC₁₋₈alkyl; protecting group, —C(O)CH-amino acid side-chain)-NHT, —NH—CH(amino acid side-chain)COOT and —CH(amino acid side-chain)COOT, where T is selected from between H and C₁₋₈alkyl; X and X′, equal or different between each other, are selected from between O and S, when a is a double bond, or X and X′ are both H, when a is a single bond, Y and Z, equal or different from each other, are selected from the group consisting of O, S, SO, SO₂ and N—R, wherein R is as above defined; Q is selected from the group consisting of C═O, CH₂, CO—NH—CH (amino acid side-chain)-CO, CONR(CH₂)_(n)CO, CONR—C₂₋₈alkenyl-CO C(O)O(CH₂)_(n)CO, CH₂OC(O)(CH₂)_(n)CO, and CH₂NRC(O)(CH₂)_(n)CO, wherein n is comprised between 2 and 6, and R is as above defined, Q′ is selected from the group consisting of C(O)OCH₂, C(O)NRCH₂, CH₂OC(O), CH₂NRC(O), CONR(CH₂)_(n)NRCO, —CONR—C₂₋₈alkenyl-NRCO, C(O)O(CH₂)_(n)NRCO, CONR(CH₂)_(n)OC(O), CH₂OC(O)(CH₂)_(n)OC(O)CH₂, CH₂NRC(O)(CH₂)_(n)NRC(O)CH₂, CH₂OC(O)(CH₂)_(n)NRC(O)CH₂, CH₂NRC(O)(CH₂)_(n)OC(O)CH₂, CH₂NR(CH₂)_(n)NRCH₂, CH₂O(CH₂)_(n)OCH₂, CH₂O(CH₂)_(n)NRCH₂, and CH₂NR(CH₂)_(n)OCH₂, wherein n is comprised between 2 and 6, and R is as above defined, and where the groups alkyl, alkenyl, alkynyl, cycloalkyl, aryl and the heterocyclic groups above reported, are possibly substituted.

Further subject of the invention are the novel 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I) and their dimers of general formula (II) and (III) above reported.

Further subject of the invention is the use of 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I) and their dimers of general formula (II) and (III) above reported for the preparation of pharmaceutical compositions useful for the treatment of:

i) neurodegenerative disorders of the Central Nervous System, such as Alzheimer Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Huntington disease, neuropathies, neural damage caused by hypoxia, ischaemia, or trauma, inducing apoptosis of nervous cells;

ii) acquired immunodeficiency diseases related reduced bioavailability of NGF, such as immunodeficiency of ageing;

iii) diseases in which stimulation of neoangiogenesis turns out to be advantageous, such as myocardial infarction, stroke, or peripheral vasculopathies;

iv) certain pathologies of the eye, such keratitis of diverse aetiology, glaucoma, degenerative or inflammatory conditions of the retina.

Further subject of the invention is the use of 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I), their dimers of general formula (II) or (III) above reported, and mixtures thereof, for the preparation of culture and storage media useful for conservation of explanted corneas destined to transplantation, and the use for promoting in vivo, in vitro, or ex vivo growth and/or survival of neural cells.

Subject of the invention is also the use of 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I), their dimers of general formula (II) or (III) above reported, and mixtures thereof, labelled with suitable reagents (contrast agents, radioisotopes, fluorescent agents etc.), and processed with any procedure useful for medical imaging purposes, for the imaging analysis of tissues and organs containing neurotrophine receptors, either in vitro or in vivo, in particular for monitoring the use and efficacy of drugs, as well as for the diagnosis of mammal diseases in which the neurothrophine receptors are involved.

The characteristic and advantages of the pharmaceutical compositions according the invention will be in detail reported in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of present compounds on PC12 cell survival in serum-free conditions, by using hrNGF as internal standard, according to paragraph “Biological Activity” hereinafter reported. Results were expressed as survival induced by compounds/spontaneous survival*100 for the compounds indicated on x axis.

FIG. 2 shows the effect of present compounds on proliferative activity of PC3 cell line, in serum-free conditions, evaluated by using hrNGF as internal standard according to paragraph “Biological Activity”. Results are expressed in terms of stimulation index, i.e. as ratio between ³H-thymidine incorporation (mean±SD) of stimulated cultures and ³H-thymidine incorporation of non stimulated cultures, for the compounds indicated on x axis.

FIG. 3 illustrates the ability of present compounds (I), (II) and (III) to induce the VGF production by PC12 cells, evaluated as hereinafter described in paragraph “Biological Activity” in comparison with hrNGF. The control is 68 Kda VGF.

FIGS. 4 a and 4 b show the ability of present compounds to displace the ¹²⁵I-NGF binding to PC12 cells, by a displacement curve obtained by analysing the resultant cell bound radioactivity in the presence of the present compounds or in the presence of hrNGF with adequate software (Graphit 4) according to paragraph “Biological Activity”.

FIG. 4 a shows the displacement curve obtained with the present compound 9 used as competitor. The analysis of data revealed a Kd of 165 nM±0.05.

FIG. 4 b shows the displacement curve obtained by using hrNGF as competitor. The analysis of data revealed a Kd of 114 pM±0.01.

FIG. 5 shows the ability of the present compounds 272, 325, 9 and 91 to induce Trk-A autophosphorylation, by using hrNGF as internal standard according to paragraph “Biological Activity”.

FIG. 6 shows the results obtained for the present compounds 9 and 325 and for the combination of the same two compounds, in a PC12 survival assay in serum-free condition, according to paragraph “Biological Activity”. The results were expressed as survival induced by compounds/spontaneous survival*100.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention by the expression “amino acid side chain”. It is meant the side chain moieties of the natural occurring L or D amino acids or of the rare or non naturally occurring amino acids.

If it is not otherwise specified, the terms alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl and heterocycle, as used in the present invention, should be meant as follows:

-   -   C₁₋₈alkyl, C₂₋₈ alkenyl and C₂₋₈ alkynyl relate to linear or         branched alkyl radicals, having only single bonds, at least one         double bond, at least one triple bond respectively. Examples of         alkylic groups according the present invention include but are         not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl,         hexyl, heptyl, octyl. Examples of alkenyl groups, according to         the present invention, include but are not limited to ethenyl,         propenyl, 1-butenyl, cis-2-butenyl, trans-2-butenyl,         2-methyl-1-propenyl, 1-pentenyl, cis-2-pentenyl,         trans-2-pentenyl, 2-methyl-2-butenyl. Examples of alkynyl groups         according to the present invention include, but are not limited         to, ethynyl, propynyl 1-butynyl, 2-butynyl, 1-pentynyl,         3-methyl-1-butynyl;     -   by the term “cycloalkyl” a ring containing carbon atom is meant,         generally having from 3 to 8 members, and preferably from 5 to 6         members. Examples of cycloalkyl groups include, but are not         limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,         cycloheptyl, cyclooctyl, norbornanyl, canphanyl, adamantanyl;     -   the term “aryl” indicates a group containing one or more         unsaturated rings, each ring having from 5 to 8 members,         preferably 5 or 6 members. Examples of aryl groups include, but         are not limited to phenyl, biphenyl and naphtyl;     -   the term “heterocycle” relates to saturated or aromatic         heterocycles containing one or more heteroatoms, and preferably         one or more N atoms. Examples of heterocycles include, but are         not limited to pyridine, imidazole, pyrrole, indole, triazoles,         pyrrolidine, pyperidine;     -   the term “arylalkyl” indicates a group having an alkyl and an         aryl substituent as above defined. As example, arylalkyl         includes but is not limited to ethylphenyl, isobutylphenyl,         benzyl, ethylbenzyl, propylbenzyl, isopropylbenzyl, butylbenzyl,         isobutylbenzyl, cycloexylbenzyl, stirenyl and biphenyl.

In the present invention the groups fluorenylmethoxycarbonyl, t-butyloxycarbonyl, carboxybenzyl, benzyl, phenyl and acetyl are indicated using the common terms Fmoc, Boc, Cbz, Bn, Ph and Ac respectively.

Preferred are the present compounds of formula (I), (II) and (III) wherein Z is O. According to the present invention the alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclic groups may be substituted with one or more moieties, and preferably one or two moieties chosen from the group consisting, of halogen, cyano, nitro, amino, hydroxy, carboxylic acid, carbonyl and C₁₋₆ alkyl. The term “halogen” relates to fluorine, chlorine, bromine and iodine.

Among the compounds of general formula (I), (II) and (III) according the invention, the specific compounds reported in the following Tables 1-4 resulted of particular interest for their agonist activity against neurotrophines, and in particular of human NGF; and thus they are the compounds preferably used for the preparation of the pharmaceutical compositions according to the invention.

TABLE 1 (I)

Compound X R₁ R₂ R₃ R₆ 1 O H H PhCH₂ (R) —CO₂Me 2 O H H PhCH₂ (S) —CO₂Me 3 O H H PhCH₂ (R) —CON(CH₂)₅ 4 O H H PhCH₂ (R) —CON(CH₂)₄ 5 O H (S) —Me PhCH₂ (R) —CO₂Me 6 O H (S) —Me PhCH₂ (S) —CO₂Me 7 O H (R) —Me PhCH₂ (R) —CO₂Me 8 O H (R) —Me PhCH₂ (S) —CO₂Me 9 O H (R) —CH₂Ph PhCH₂ (S) —CO₂Me 10 O H (R) —CH₂Ph PhCH₂ (R) —CO₂Me 11 O H (S) —CH₂Ph PhCH₂ (S) —CO₂Me 12 O H (S) —CH₂Ph PhCH₂ (R) —CO₂Me 13 O H (S) —CH₂OBn PhCH₂ (R) —CO₂Me 14 O H (S) —CH₂OBn PhCH₂ (S) —CO₂Me 15 O H (R) —CH₂OBn PhCH₂ (R) —CO₂Me 16 O H (R) —CH₂OBn PhCH₂ (S) —CO₂Me 17 O H (S) —CH₂OH PhCH₂ (R) —CO₂Me 18 O H (S) —CH₂OH PhCH₂ (S) —CO₂Me 19 O H (R) —CH₂OH PhCH₂ (R) —CO₂Me 20 O H (R) —CH₂OH PhCH₂ (S) —CO₂Me 21 O H ═CH₂ PhCH₂ (R) —CO₂Me 22 O H ═CH₂ PhCH₂ (S) —CO₂Me 23 O H (R) —CH₂OH PhCH₂ (S) —CO₂Me 24 S H H PhCH₂ (R) —CO₂Me 25 S H H PhCH₂ (R) —CONH(CH₂)₂NH₂ 26 S H H PhCH₂ (R) —CONH(CH₂)₂OH 27 O Ph H PhCH₂ (R) —CO₂Me 28 O Ph H PhCH₂ (S) —CO₂Me 29 O Ph H CH(Ph)₂ (R) —CO₂Me 30 O Ph H CH(Ph)₂ (S) —CO₂Me 31 O NO₂-Ph H Ph (S)—CO₂Me 32 H H H H (R) —CO₂H 33 H H H H (S) —CO₂H 34 H H H H (R) —CO₂Me 35 H H H H (S) —CO₂Me 36 H H H PhCH₂ (R) —CO₂H 37 H H H PhCH₂ (S) —CO₂H 38 H H H Fmoc (R) —CO₂H 39 H H H Fmoc (S) —CO₂H 40 H H H PhCH₂ (R) —CO₂Me 41 H H H PhCH₂ (S) —CO₂Me 42 H H H Boc (R) —CO₂Me 43 H H H Boc (S) —CO₂Me 44 H H H Fmoc (R) —CO₂Me 45 H H H Fmoc (S) —CO₂Me 46 H H H H (R) —CONHMe 47 H H H H (S) —CONHMe 48 H H H Ac (R) —CONHMe 49 H H H Ac (S) —CONHMe 50 H H H PhCH₂ (R) —CONHMe 51 H H H PhCH₂ (S) —CONHMe 52 H H H Fmoc (R) —CONHMe 53 H H H Fmoc (S) —CONHMe 54 H H H PhCH₂ (R) —CON(CH₂)₅ 55 H H H PhCH₂ (R) —CONHcyclohexyl 56 H H H PhCH₂ (R) —CON(CH₂)₄ 57 H H H PhCH₂ (R) —CONH(CH₂)₂OH 58 H H H H (R) —CH₂OH 59 H H H H (S) —CH₂OH 60 H H H Fmoc (S) —CH₂OH 61 H H H Fmoc (R) —CH₂OH 62 H H H Boc (R) —CH₂OH 63 H H H Boc (S) —CH₂OH 64 H H H PhCH₂ (R) —CH₂OH 65 H H H PhCH₂ (S) —CH₂OH 66 H H (S) —CH₂OBn PhCH₂ (R) —CO₂Me 67 H H (S) —CH₂OBn PhCH₂ (S) —CO₂Me 68 H H (R) —CH₂OBn PhCH₂ (R) —CO₂Me 69 H H (R) —CH₂OBn PhCH₂ (S) —CO₂Me 70 H H (S) —CH₂OBn PhCH₂ (R) —CH₂OH 71 H H (S) —CH₂OBn PhCH₂ (S) —CH₂OH 72 H H (R) —CH₂OBn PhCH₂ (R) —CH₂OH 73 H H (R) —CH₂OBn PhCH₂ (S) —CO₂Me 75 H H (S) —COOH Fmoc (R) —CO₂Me 76 H H (S) —COOH Fmoc (S) —CO₂Me 77 H H (R) —COOH Fmoc (R) —CO₂Me 78 H H (R) —COOH Fmoc (S) —CO₂Me 79 H H (S) —CH₂OBn Fmoc (R) —CO₂Me 80 H H (S) —CH₂OBn Fmoc (S) —CO₂Me 81 H H (R) —CH₂OBn Fmoc (R) —CO₂Me 82 H H (R) —CH₂OBn Fmoc (S) —CO₂Me 83 H H (S) —CH₂OBn H (R) —CO₂Me 84 H H (S) —CH₂OBn H (S) —CO₂Me 85 H H (R) —CH₂OBn H (R) —CO₂Me 86 H H (R) —CH₂OBn H (S) —CO₂Me 87 H H (S) —CH₂OH H (R) —CO₂Me 88 H H (S) —CH₂OH H (S) —CO₂Me 89 H H (R) —CH₂OH H (R) —CO₂Me 90 H H (R) —CH₂OH H (S) —CO₂Me 91 H H (S) —CH₂OH Fmoc (R) —CO₂Me 92 H H (S) —CH₂OH Fmoc (S) —CO₂Me 93 H H (R) —CH₂OH Fmoc (R) —CO₂Me 94 H H (R) —CH₂OH Fmoc (S) —CO₂Me 95 H H (S) —CH₂OH Fmoc (R) —CO₂Me 96 H H (S) —CH₂OH Fmoc (S) —CO₂Me 97 H H (R) —CH₂OH Fmoc (R) —CO₂Me 98 H H (R) —CH₂OH Fmoc (S) —CO₂Me 99 H H (S) —CH₂OH PhCH₂ (S) —CO₂Me 100 H H (R) —CH₂OH PhCH₂ (R) —CO₂Me 101 H H (R) —CH₂OH PhCH₂ (R) —CO₂Me 102 H H (R) —CH₂OH PhCH₂ (S) —CO₂Me 103 H H (S) —CH₂OH Fmoc (R) —CO₂OH 104 H H (S) —CH₂OH Fmoc (S) —CO₂OH 105 H H (R) —CH₂OH Fmoc (R) —CO₂OH 106 H H (R) —CH₂OH Fmoc (S) —CO₂OH 107 H H (S) —CH₂OH PhCH₂ (R) —CO₂OH 108 H H (S) —CH₂OH PhCH₂ (S) —CO₂OH 109 H H (R) —CH₂OH PhCH₂ (R) —CO₂OH 110 H H (R) —CH₂OH PhCH₂ (S) —CO₂OH 111 H H ═CH₂ PhCH₂ (R) —CO₂Me 112 H H ═CH₂ PhCH₂ (S) —CO₂Me 113 H H ═CH₂ PhCH₂ (R) —CH₂OH 114 H H ═CH₂ PhCH₂ (S) —CH₂OH 115 H H (S) —CH₂CH(Me)₂ Fmoc (R) —CH₂OH 116 H H (S) —CH₂CH(Me)₂ PhCH₂ (S) —CH₂OH 117 H H (S) —CH₂CH(Me)₂ H (R) —CH₂OH 118 H Ph H H (R) —CO₂Me 119 H Ph H Fmoc (R) —CO₂Me 120 H Ph H PhCH₂ (R) —CO₂Me 121 H Ph H CH(Ph)₂ (R) —CO₂Me 122 H Ph H H (S) —CO₂Me 123 H Ph H Fmoc (S) —CO₂Me 124 H Ph H PhCH₂ (S) —CO₂Me 125 H Ph H CH(Ph)₂ (S) —CO₂Me 126 H p-NH₂—C₆H₄ H Ph (S) —COOMe 127 H p-NH₂—C₆H₄ H Ph (S) —COOH 128 H p-NH₂—C₆H₄ H Ph (S) —CONHCH₂CO₂Me 129 H p-NH—(Asp(O^(t)Bu)— H Ph (S) —CO₂Me NH₂) C₆H₄ 130 H p-NH—(Asp(O^(t)Bu)— H Ph (S) —CO₂H NH₂)—C₆H₄ 131 H p-NH—(Asp(O^(t)Bu)— H Ph (S) —CONH—Lys NH₂) C₆H₄ (NHBoc)—OMe 132 H p-NH—(Asp(OH)— H Ph (S) —CONH—Lys—OMe NH₂)—C₆H₄ 133 H p-NO₂—C₆H₄ H Ph (S) —COOH 134 H p-NO₂—C₆H₄ H Ph (S) —COOMe 135 H p-NO₂—C₆H₄ H Ph (S) —CONHCH₂CO₂Me 136 H Ph H H (R) —CH₂OH 137 H Ph H Fmoc (R) —CH₂OH 138 H Ph H PhCH₂ (R) —CH₂OH 139 H Ph H CH(Ph)₂ (R) —CH₂OH 140 H Ph H H (S) —CH₂OH 141 H Ph H Fmoc (S) —CH₂OH 142 H Ph H PhCH₂ (S) —CH₂OH 143 H Ph H CH(Ph)₂ (S) —CH₂OH 144 H H (S) —Me Fmoc (R) —CO₂OH 145 H H (S) —Me Fmoc (S) —CO₂OH 146 H H (R) —Me Fmoc (R) —CO₂OH 147 H H (R) —Me Fmoc (S) —CO₂OH 148 H H (S) —Me Fmoc (R) —CO₂Me 149 H H (S) —Me Fmoc (S) —CO₂Me 150 H H (R) —Me Fmoc (R) —CO₂Me 151 H H (R) —Me Fmoc (S) —CO₂Me 152 H H (S) —Me PhCH₂ (R) —CO₂Me 153 H H (S) —Me PhCH₂ (S) —CO₂Me 154 H H (R) —Me PhCH₂ (R) —CO₂Me 155 H H (R) —Me PhCH₂ (S) —CO₂Me 156 H H (S) —Me Fmoc (R) —CH₂OH 157 H H (S) —Me Fmoc (S) —CH₂OH 158 H H (R) —Me Fmoc (R) —CH₂OH 159 H H (S) —Me Fmoc (S) —CH₂OH 160 H H (S) —Me PhCH₂ (R) —CH₂OH 161 H H (S) —Me PhCH₂ (S) —CH₂OH 162 H H (R) —Me PhCH₂ (R) —CH₂OH 163 H H (R) —Me PhCH₂ (S) —CH₂OH 164 H H (S) —PhCH₂ Fmoc (R) —CO₂H 165 H H (S) —PhCH₂ Fmoc (S) —CO₂H 166 H H (R) —PhCH₂ Fmoc (R) —CO₂H 167 H H (R) —PhCH₂ Fmoc (S) —CO₂H 168 H H (S) —PhCH₂ Fmoc (R) —CO₂Me 169 H H (S) —PhCH₂ Fmoc (S) —CO₂Me 170 H H (R) —PhCH₂ Fmoc (R) —CO₂Me 171 H H (R) —PhCH₂ Fmoc (R) —CO₂Me 172 H H (S) —PhCH₂ PhCH₂ (R) —CO₂Me 173 H H (S) —PhCH₂ PhCH₂ (S) —CO₂Me 174 H H (R) —PhCH₂ PhCH₂ (R) —CO₂Me 175 H H (R) —PhCH₂ PhCH₂ (S) —CO₂Me 176 H H (R) —PhCH₂ H (R) —CO₂Me 177 H H (R) —PhCH₂ H (S) —CO₂Me 178 H H (S) —PhCH₂ H (R) —CO₂Me 179 H H (S) —PhCH₂ H (S) —CO₂Me 180 H H (S) —PhCH₂ Fmoc (R) —CH₂OH 181 H H (S) —PhCH₂ Fmoc (S) —CH₂OH 182 H H (R) —PhCH₂ Fmoc (R) —CH₂OH 183 H H (R) —PhCH₂ Fmoc (S) —CH₂OH 184 H H (S) —PhCH₂ PhCH₂ (R) —CH₂OH 185 H H (S) —PhCH₂ PhCH₂ (S) —CH₂OH 186 H H (R) —PhCH₂ PhCH₂ (R) —CH₂OH 187 H H (R) —PhCH₂ PhCH₂ (S) —CH₂OH 188 H H (S) —PhCH₂ PhCH₂ (R)—COOH 189 O p-NO₂Ph H Ph (R)—CONH(CH₂)₆NH₂

TABLE 2 (I)

Compound X R₁ R₂ R₃ R₆ 190 O H H PhCH₂ (R) —CO₂Me 191 O H H PhCH₂ (S) —CO₂Me 192 O H (S) —Me PhCH₂ (R) —CO₂Me 193 O H (S) —Me PhCH₂ (S) —CO₂Me 194 O H (R) —Me PhCH₂ (R) —CO₂Me 195 O H (R) —Me PhCH₂ (S) —CO₂Me 196 O H (S) —PhCH₂ PhCH₂ (R) —CO₂Me 197 O H (S) —PhCH₂ PhCH₂ (S) —CO₂Me 198 O H (R) —PhCH₂ PhCH₂ (R) —CO₂Me 199 O H (R) —PhCH₂ PhCH₂ (S) —CO₂Me 200 O H (S) —CH₂CH(Me)₂ PhCH₂ (R) —CO₂Me 201 O H (S) —CH₂CH(Me)₂ PhCH₂ (S) —CO₂Me 202 O H (R) —CH₂CH(Me)₂ PhCH₂ (R) —CO₂Me 203 O H (R) —CH₂CH(Me)₂ PhCH₂ (S) —CO₂Me 204 O H H PhCH₂ (R) —CONHMe 205 O H H PhCH₂ (S) —CONHMe 206 O H (S) —Me PhCH₂ (R) —CONHMe 207 O H (S) —Me PhCH₂ (S) —CONHMe 208 O H (R) —Me PhCH₂ (R) —CONHMe 209 O H (R) —Me PhCH₂ (S) —CONHMe 210 O H (S) —PhCH₂ PhCH₂ (R) —CONHMe 211 O H (S) —PhCH₂ PhCH₂ (S) —CONHMe 212 O H (R) —PhCH₂ PhCH₂ (R) —CONHMe 213 O H (R) —PhCH₂ PhCH₂ (S) —CONHMe 214 O H (S) —CH₂CH(Me)₂ PhCH₂ (R) —CONHMe 215 O H (S) —CH₂CH(Me)₂ PhCH₂ (S) —CONHMe 216 O H (R) —CH₂CH(Me)₂ PhCH₂ (R) —CONHMe 217 O H (R) —CH₂CH(Me)₂ PhCH₂ (S) —CONHMe 218 H H H Fmoc (R) —CO₂H 219 H H H Fmoc (R) —CO₂Me 220 H H H Fmoc (S) —CO₂H 221 H H H Fmoc (S) —CO₂Me 222 H H (S) —Me Fmoc (R) —CO₂H 223 H H (S) —Me Fmoc (R) —CO₂Me 224 H H (S) —Me PhCH₂ (R) —CO₂Me 225 H H (R) —Me Fmoc (R) —CO₂H 226 H H (R) —Me Fmoc (R) —CO₂Me 227 H H (R) —Me PhCH₂ (R) —CO₂Me 228 H H (S) —Me Fmoc (S) —CO₂H 229 H H (S) —Me Fmoc (S) —CO₂Me 230 H H (S) —Me PhCH₂ (S) —CO₂Me 231 H H (R) —Me Fmoc (S) —CO₂H 232 H H (R) —Me Fmoc (S) —CO₂Me 233 H H (R) —Me PhCH₂ (S) —CO₂Me 234 H H (S) —PhCH₂ Fmoc (R) —CO₂H 235 H H (S) —PhCH₂ Fmoc (R) —CO₂Me 236 H H (S) —PhCH₂ PhCH₂ (R) —CO₂Me 237 H H (R) —PhCH₂ Fmoc (R) —CO₂H 238 H H (R) —PhCH₂ Fmoc (R) —CO₂Me 239 H H (R) —PhCH₂ PhCH₂ (R) —CO₂Me 240 H H (S) —PhCH₂ Fmoc (S) —CO₂H 241 H H (S) —PhCH₂ Fmoc (S) —CO₂Me 242 H H (S) —PhCH₂ PhCH₂ (S) —CO₂Me 243 H H (R) —PhCH₂ Fmoc (S) —CO₂H 244 H H (R) —PhCH₂ Fmoc (S) —CO₂Me 245 H H (R) —PhCH₂ PhCH₂ (S) —CO₂Me 246 H H (R) —CH₂OH Fmoc (S) —CO₂Me 247 H H (R) —CH₂OH PhCH₂ (S) —CO₂Me 248 H H (R) —CH₂OBn Fmoc (S) —CO₂Me 249 H H (R) —CH₂OBn PhCH₂ (S) —CO₂Me 250 H H (R) —CH₂OH Fmoc (R) —CO₂Me 251 H H (R) —CH₂OH PhCH₂ (R) —CO₂Me 252 H H (R) —CH₂OBn Fmoc (R) —CO₂Me 253 H H (R) —CH₂OBn PhCH₂ (R) —CO₂Me 254 H H (S) —CH₂OH Fmoc (S) —CO₂Me 255 H H (S) —CH₂OH PhCH₂ (S) —CO₂Me 256 H H (S) —CH₂OBn Fmoc (S) —CO₂Me 257 H H (S) —CH₂OBn PhCH₂ (S) —CO₂Me 258 H H (S) —CH₂OH Fmoc (R) —CO₂Me 259 H H (S) —CH₂OH PhCH₂ (R) —CO₂Me 260 H H (S) —CH₂OBn Fmoc (R) —CO₂Me 261 H H (S) —CH₂OBn PhCH₂ (R) —CO₂Me 262 H H (S) —CH₂CH(Me)₂ Bn (R) —CO₂Me 263 H H (R) —CH₂CH(Me)₂ Bn (R) —CO₂Me 264 H H (S) —CH₂CH(Me)₂ Bn (S) —CO₂Me 265 H H (R) —CH₂CH(Me)₂ Bn (S) —CO₂Me 266 H H (S) —CH₂CH(Me)₂ Fmoc (R) —CO₂Me 267 H H (R) —CH₂CH(Me)₂ Fmoc (R) —CO₂Me 268 H H (S) —CH₂CH(Me)₂ Fmoc (S) —CO₂Me 269 H H (R) —CH₂CH(Me)₂ Fmoc (S) —CO₂Me 270 H H (S) —Me H (R) —CH₂OH 271 H H (S) —Me Bn (R) —CH₂OH 272 H H (S) —Me Fmoc (R) —CH₂OH 273 H H (R) —Me H (R) —CH₂OH 274 H H (R) —Me Bn (R) —CH₂OH 275 H H (R) —Me Fmoc (R) —CH₂OH 276 H H (S) —Me H (S) —CH₂OH 277 H H (S) —Me Bn (S) —CH₂OH 278 H H (S) —Me Fmoc (S) —CH₂OH 279 H H (R) —Me H (S) —CH₂OH 280 H H (R) —Me Bn (S) —CH₂OH 281 H H (R) —Me Fmoc (S) —CH₂OH 282 H H (S) —CH₂CH(Me)₂ H (R) —CH₂OH 283 H H (S) —CH₂CH(Me)₂ Bn (R) —CH₂OH 284 H H (S) —CH₂CH(Me)₂ Fmoc (R) —CH₂OH 285 H H (R) —CH₂CH(Me)₂ H (R) —CH₂OH 286 H H (R) —CH₂CH(Me)₂ Bn (R) —CH₂OH 287 H H (R) —CH₂CH(Me)₂ Fmoc (R) —CH₂OH 288 H H (S) —CH₂CH(Me)₂ H (S) —CH₂OH 289 H H (S) —CH₂CH(Me)₂ Bn (S) —CH₂OH 290 H H (S) —CH₂CH(Me)₂ Fmoc (S) —CH₂OH 291 H H (R) —CH₂CH(Me)₂ H (S) —CH₂OH 292 H H (R) —CH₂CH(Me)₂ Bn (S) —CH₂OH 293 H H (R) —CH₂CH(Me)₂ Fmoc (S) —CH₂OH 294 H H (S) —PhCH₂ H (R) —CH₂OH 295 H H (S) —PhCH₂ Bn (R) —CH₂OH 296 H H (S) —PhCH₂ Fmoc (R) —CH₂OH 297 H H (R) —PhCH₂ H (R) —CH₂OH 298 H H (R) —PhCH₂ Bn (R) —CH₂OH 299 H H (R) —PhCH₂ Fmoc (R) —CH₂OH 300 H H (S) —PhCH₂ H (S) —CH₂OH 301 H H (S) —PhCH₂ Bn (S) —CH₂OH 302 H H (S) —PhCH₂ Fmoc (S) —CH₂OH 303 H H (R) —PhCH₂ H (S) —CH₂OH 304 H H (R) —PhCH₂ Bn (S) —CH₂OH 305 H H (R) —PhCH₂ Fmoc (S) —CH₂OH 306 H H (R) —CH₂OH Fmoc (S) —CH₂OH 307 H H (R) —CH₂OH PhCH₂ (S) —CH₂OH 308 H H (R) —CH₂OBn Fmoc (S) —CH₂OH 309 H H (R) —CH₂OBn PhCH₂ (S) —CH₂OH 310 H H (R) —CH₂OH Fmoc (R) —CH₂OH 311 H H (R) —CH₂OH PhCH₂ (R) —CH₂OH 312 H H (R) —CH₂OBn Fmoc (R) —CH₂OH 313 H H (R) —CH₂OBn PhCH₂ (R) —CH₂OH 314 H H (S) —CH₂OH Fmoc (S) —CH₂OH 315 H H (S) —CH₂OH PhCH₂ (S) —CH₂OH 316 H H (S) —CH₂OBn Fmoc (S) —CH₂OH 317 H H (S) —CH₂OBn PhCH₂ (S) —CH₂OH 318 H H (S) —CH₂OH Fmoc (R) —CH₂OH 319 H H (S) —CH₂OH PhCH₂ (R) —CH₂OH 320 H H (S) —CH₂OBn Fmoc (R) —CH₂OH 321 H H (S) —CH₂OBn PhCH₂ (R) —CH₂OH

TABLE 3 (II)

Compound R₁ R₂ R₃ R′₁ R′₂ R₆ 322 H H H H H CO₂Me 323 H H H H H CONHMe 324 H H PhCH₂ H H CO₂Me 325 H H PhCH₂ H H CONHMe 326 H H Fmoc H H CO₂Me 327 H H Fmoc H H CONHMe 328 H H Boc H H CO₂Me 329 H H Boc H H CONHMe 330 H PhCH₂ H H H CO₂Me 331 H PhCH₂ H H H CONHMe 332 H PhCH₂ PhCH₂ H H CO₂Me 333 H PhCH₂ PhCH₂ H H CONHMe 334 H PhCH₂ Fmoc H H CO₂Me 335 H PhCH₂ Fmoc H H CONHMe 336 H PhCH₂ Boc H H CO₂Me 337 H PhCH₂ Boc H H CONHMe 338 H H H H PhCH₂ CO₂Me 339 H H H H PhCH₂ CONHMe 340 H H PhCH₂ H PhCH₂ CO₂Me 341 H H PhCH₂ H PhCH₂ CONHMe 342 H H Fmoc H PhCH₂ CO₂Me 343 H H Fmoc H PhCH₂ CONHMe 344 H H Boc H PhCH₂ CO₂Me 345 H H Boc H PhCH₂ CONHMe 346 H PhCH₂ H H PhCH₂ CO₂Me 347 H PhCH₂ H H PhCH₂ CONHMe 348 H PhCH₂ PhCH₂ H PhCH₂ CO₂Me 349 H PhCH₂ PhCH₂ H PhCH₂ CONHMe 350 H PhCH₂ Fmoc H PhCH₂ CO₂Me 351 H PhCH₂ Fmoc H PhCH₂ CONHMe 352 H PhCH₂ Boc H PhCH₂ CO₂Me 353 H PhCH₂ Boc H PhCH₂ CONHMe 354 Ph H H H H CO₂Me 355 Ph H H H H CONHMe 356 Ph H PhCH₂ H H CO₂Me 357 Ph H PhCH₂ H H CONHMe 358 Ph H Fmoc H H CO₂Me 359 Ph H Fmoc H H CONHMe 360 Ph H Boc H H CO₂Me 361 Ph H Boc H H CONHMe 362 H H H Ph H CO₂Me 363 H H H Ph H CONHMe 364 H H PhCH₂ Ph H CO₂Me 365 H H PhCH₂ Ph H CONHMe 366 H H Fmoc Ph H CO₂Me 367 H H Fmoc Ph H CONHMe 368 H H Boc Ph H CO₂Me 369 H H Boc Ph H CONHMe 370 Ph H H Ph H CO₂Me 371 Ph H H Ph H CONHMe 372 Ph H PhCH₂ Ph H CO₂Me 373 Ph H PhCH₂ Ph H CONHMe 374 Ph H Fmoc Ph H CO₂Me 375 Ph H Fmoc Ph H CONHMe 376 Ph H Boc Ph H CO₂Me 377 Ph H Boc Ph H CONHMe 378 H H H H CH₂OH CO₂Me 379 H H H H CH₂OH CONHMe 380 H H PhCH₂ H CH₂OH CO₂Me 381 H H PhCH₂ H CH₂OH CONHMe 382 H H Fmoc H CH₂OH CO₂Me 383 H H Fmoc H CH₂OH CONHMe 384 H H Boc H CH₂OH CO₂Me 385 H H Boc H CH₂OH CONHMe 386 H PhCH₂ H H CH₂OH CO₂Me 387 H PhCH₂ H H CH₂OH CONHMe 388 H PhCH₂ PhCH₂ H CH₂OH CO₂Me 389 H PhCH₂ PhCH₂ H CH₂OH CONHMe 390 H PhCH₂ Fmoc H CH₂OH CO₂Me 391 H PhCH₂ Fmoc H CH₂OH CONHMe 392 H PhCH₂ Boc H CH₂OH CO₂Me 393 H PhCH₂ Boc H CH₂OH CONHMe 394 Ph H H H CH₂OH CO₂Me 395 Ph H H H CH₂OH CONHMe 396 Ph H PhCH₂ H CH₂OH CO₂Me 397 Ph H PhCH₂ H CH₂OH CONHMe 398 Ph H Fmoc H CH₂OH CO₂Me 399 Ph H Fmoc H CH₂OH CONHMe 400 Ph H Boc H CH₂OH CO₂Me 401 Ph H Boc H CH₂OH CONHMe

TABLE 4 (III)

Compound R₁ R₂ R₃ R′₁ R′₂ R₃ X Q′ 402 H H H H H H O CO—NH(CH₂)₂NH—CO 403 H H H H H H O CO—NH(CH₂)₄NH—CO 404 H H H H H H O CO—NH(CH₂)₆NH—CO 405 H H H H H H O CO—N(C₂H₄)N—CO 406 H H PhCH₂ H H PhCH₂ O CO—NH(CH₂)₂NH—CO 407 H H PhCH₂ H H PhCH₂ O CO—NH(CH₂)₄NH—CO 408 H H PhCH₂ H H PhCH₂ O CO—NH(CH₂)₆NH—CO 409 H H PhCH₂ H H PhCH₂ O CO—N(C₂H₄)N—CO 410 H H PhCH₂ H H PhCH₂ H CO—NH(CH₂)₂NH—CO 411 H H PhCH₂ H H PhCH₂ H CO—NH(CH₂)₄NH—CO 412 H H PhCH₂ H H PhCH₂ H CO—NH(CH₂)₆NH—CO 413 H H PhCH₂ H H PhCH₂ H CO—N(C₂H₄)N—CO 414 H PhCH₂ PhCH₂ H PhCH₂ PhCH₂ O CO—NH(CH₂)₂NH—CO 415 H PhCH₂ PhCH₂ H PhCH₂ PhCH₂ O CO—NH(CH₂)₄NH—CO 416 H PhCH₂ PhCH₂ H PhCH₂ PhCH₂ O CO—NH(CH₂)₆NH—CO 417 H PhCH₂ PhCH₂ H PhCH₂ PhCH₂ O CO—N(C₂H₄)N—CO 418 H PhCH₂ PhCH₂ H PhCH₂ PhCH₂ H CO—NH(CH₂)₂NH—CO 419 H PhCH₂ PhCH₂ H PhCH₂ PhCH₂ H CO—NH(CH₂)₄NH—CO 420 H PhCH₂ PhCH₂ H PhCH₂ PhCH₂ H CO—NH(CH₂)₆NH—CO 421 H PhCH₂ PhCH₂ H PhCH₂ PhCH₂ H CO—N(C₂H₄)N—CO 422 Ph H PhCH₂ Ph H PhCH₂ O CO—NH(CH₂)₂NH—CO 423 Ph H PhCH₂ Ph H PhCH₂ O CO—NH(CH₂)₄NH—CO 424 Ph H PhCH₂ Ph H PhCH₂ O CO—NH(CH₂)₆NH—CO 425 Ph H PhCH₂ Ph H PhCH₂ O CO—N(C₂H₄)N—CO 426 Ph H PhCH₂ Ph H PhCH₂ H CO—NH(CH₂)₂NH—CO 427 Ph H PhCH₂ Ph H PhCH₂ H CO—NH(CH₂)₄NH—CO 428 Ph H PhCH₂ Ph H PhCH₂ H CO—NH(CH₂)₆NH—CO 429 Ph H PhCH₂ Ph H PhCH₂ H CO—N(C₂H₄)N—CO 430 Ph H PhCH₂ Ph H PhCH₂ H CO—NH(CH₂)₂NH—CO 431 Ph H PhCH₂ Ph H PhCH₂ H CO—NH(CH₂)₄NH—CO 432 Ph H PhCH₂ Ph H PhCH₂ H CO—NH(CH₂)₆NH—CO 433 Ph H PhCH₂ Ph H PhCH₂ H CO—N(C₂H₄)N—CO 434 Ph H Ph Ph H Ph O CO—NH(CH₂)₂NH—CO 435 Ph H Ph Ph H Ph O CO—NH(CH₂)₄NH—CO 436 Ph H Ph Ph H Ph O CO—NH(CH₂)₆NH—CO 437 Ph H Ph Ph H Ph O CO—N(C₂H₄)N—CO 438 NO₂—Ph H Ph NO₂—Ph H Ph O CO—NH(CH₂)₂NH—CO 439 NO₂—Ph H Ph NO₂—Ph H Ph O CO—NH(CH₂)₃NH—CO 440 NO₂—Ph H Ph NO₂—Ph H Ph O CO—NH(CH₂)₄NH—CO 441 NO₂—Ph H Ph NO₂—Ph H Ph O CO—NH(CH₂)₅NH—CO 442 NO₂—Ph H Ph NO₂—Ph H Ph O CO—NH(CH₂)₆NH—CO 443 NO₂—Ph H Ph NH₂—Ph H Ph O CO—N(C₂H₄)N—CO 444 NH₂—Ph H Ph NH₂—Ph H Ph O CO—NH(CH₂)₂NH—CO 445 NH₂—Ph H Ph NH₂—Ph H Ph O CO—NH(CH₂)₃NH—CO 446 NH₂—Ph H Ph NH₂—Ph H Ph O CO—NH(CH₂)₄NH—CO 447 NH₂—Ph H Ph NH₂—Ph H Ph O CO—NH(CH₂)₅NH—CO 448 NH₂—Ph H Ph NH₂—Ph H Ph O CO—NH(CH₂)₆NH—CO 449 NH₂—Ph H Ph NH₂—Ph H Ph O CO—N(C₂H₄)N—CO 450 NO₂—Ph H Ph NO₂—Ph H Ph H CO—NH(CH₂)₂NH—CO 451 NO₂—Ph H Ph NO₂—Ph H Ph H CO—NH(CH₂)₃NH—CO 452 NO₂—Ph H Ph NO₂—Ph H Ph H CO—NH(CH₂)₄NH—CO 453 NO₂—Ph H Ph NO₂—Ph H Ph H CO—NH(CH₂)₅NH—CO 454 NO₂—Ph H Ph NO₂—Ph H Ph H CO—NH(CH₂)₆NH—CO 455 NO₂—Ph H Ph NH₂—Ph H Ph H CO—N(C₂H₄)N—CO 456 NH₂—Ph H Ph NH₂—Ph H Ph H CO—NH(CH₂)₂NH—CO 457 NH₂—Ph H Ph NH₂—Ph H Ph H CO—NH(CH₂)₃NH—CO 458 NH₂—Ph H Ph NH₂—Ph H Ph H CO—NH(CH₂)₄NH—CO 459 NH₂—Ph H Ph NH₂—Ph H Ph H CO—NH(CH₂)₅NH—CO 460 NH₂—Ph H Ph NH₂—Ph H Ph H CO—NH(CH₂)₆NH—CO 461 NH₂—Ph H Ph NH₂—Ph H Ph H CO—N(C₂H₄)N—CO

In particular, as far as the dimers of formula (II) and (III) are concerned, all the possible combinations of the stereoisomers are possible, although not exactly specified in the above Table 3 and 4.

Furthermore, the present invention refers to the derivatives of 3-aza-bicyclo[3.2.1]octanes and their dimers that were prepared by the Applicants and described here for the first time, i.e. the 3-aza-bicyclo[3.2.1]octane derivatives (I) and their dimers of general formula (II) and (III) defined as above with exclusion of the following compounds: 1, 2, 5, 7, 8, 9, 10, 12, 13, 17, 19, 20, 21, 32, 34, 35, 36, 38, 40, 44, 58, 60, 64, 65, 66, 70, 75, 76, 77, 78, 79 83, 87, 91, 95, 99, 101, 103, 138, 145, 152, 154, 163, 164, 168, 172, 174, 176, 178, 184, 186, 192, 322, 324.

The compounds above cited are indeed already described in J. Org. Chem. 1999, 64, 7347, Organic Letters, 2000, 2, 3987-3990, Bioorganic & Med Chem 2001, 9, 1625-1632, Eur. J. Org. Chem. 2002, 873-880, and in the European Application Patent No. 00104135.9-2117 and in the International Application No. WO 01/64686; in such documents the preparation methods of the compounds are also described.

The novel derivatives of 3-aza-bicyclo[3.2.1]octanes of general formula (I) and their dimers of general formula (II) and (III) may be prepared with the following process. The new compounds of general formula (I) and their correspondent dimers of formula (II) and (III), described for the first time in the present application may be prepared according the procedure described as following and represented in the following Scheme 1:

Protected alpha amino aldehydes (3a) or alpha amino ketones (3b) or alpha amino alcohols (3c) were reacted with—activated derivatives of tartaric acid as for example diacetyloxytartaric anydride 4 (R,R or S,S),—or with acid tartaric derivatives as for example the protected mono-methylester 6 (R,R or S,S), in the presence of coupling and activating agents—or by reductive amination with protected derivatives of erithrolactole 5 (R, R prepared from D-arabinose or S,S prepared from L-arabinose). The correspondent amides 7 e 9 (in the scheme 1 are shown only the R, R enantiomers, but the enantiomers S,S were prepared analogously) or amine 8 (in the scheme 1 are shown only the R,S enantiomers, but the S,R enantiomers were prepared analogously). In the case of amide alcohol 9 the correspondent aldehyde or ketone 10 are obtained by oxidation. When R₃ is H in the amine 8, a Fmoc protection can be made. The further cyclisation of compounds 7, 8 e 10 (Scheme 1) occurs by treatment with SOCl₂ and MeOH (reaction condition i) followed by treatment with sulfuric acid adsorbed on SiO₂ in refluxing toluene (reaction conditions ii) or by treatment with trifluoro acetic acid (TFA) pure or in methylene chloride (reaction conditions iii). Thus, starting from amides 7 and 10, the compounds I wherein X=O and R₆=—COOMe in configuration exo were prepared. In the case of amine 8 compounds I, wherein X=H, H and the group R₆=—CH₂OH in endo configuration were prepared. The configuration R,R or S,S of stereocenters at C-1 bridgehead and at C-7 (bearing the carboxylic or hydroxymethyl group) is depending from that of tartaric acid or from starting erithrolactole. The compounds I may be modified according to Scheme 2.

using the complex BH₃ dimethyl sulfide, either to correspondent amino esters I (X=H, H, R₆=COOMe), or to correspondent amino alcohol I (X=H, H e R₆=CH₂OH). Such compounds may be deprotected to nitrogen atom. The hydrolysis of amino ester I (X=H, H, R=COOMe) may be done either in acid or basic conditions, giving to the correspondent amino acid I (X=H, H e R₆=COOH). The amino acid is also obtained by Jones oxidation or by using PDC in DMF, from amino alcohol I (X=H, H e R₆=CH₂OH), also after the change of the benzyl group to Boc or Fmoc. By activation of the carboxylic group an amide bond with an amine NHR₇R₈ or an amino acid is formed. Otherwise, the activated carboxylic group of the amino acid I, is reacted with another unit of I having the deprotected nitrogen, to give the dimers of general formula (II) present in Table 3.

Otherwise, two units of a compound of formula (I) in each form, is reacted with a spacer Q, to give the dimers of general formula (III). The example shown in the scheme 2 includes but is not limited to the reaction of a diamine (Q) with two units of an activated carboxylic acid to give dimers of formula (III) reported in Table 4. The present 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I) and their dimers of general formula (II) and (III), in free form or in form of pharmaceutically acceptable salts, may be used for preparation of pharmaceutical compositions following usual methods of pharmaceutical preparation.

Such pharmaceutical compositions may be formulated in conventional way, and may include one or more eccipients and/or diluent pharmaceutically acceptable. Administration of such formulations is feasible through any conventional route, such as parenteral, in the form of solution or suspension, oral, ocular, nasal, topical, etc.

The formulation of the 3-aza-bicyclo[3.2.1]octane derivatives of formula (I) and of their dimers of formula (II) and (III) according to the invention include tablets, capsules, pills, pellets, solutions, dispersions, suspensions, liposomal formulations, microspheres, nanospheres, creams and ointments, emulsions and aerosols, that can also be prepared in a way that allows a controlled or retarded release of the active compound.

Such pharmaceutical compositions may comprise at least one among the present compounds of formula (I), (II) and (III), or mixtures thereof, as active principle, possibly even in combination with other active principle or co-adjuvant, selected according to the pathologic conditions.

The pharmaceutical compositions comprising the compounds of the invention are suitable for pharmaceutical treatment of pathologic conditions related to the activity of neurotrophins.

The present derivatives of 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I) and their dimers of general formula (II) showed neurotrophin agonist activity, especially of NGF, as they have the property of interacting with the NGF receptor complex at defined affinity levels. The agonist compounds have the property of inducing the biological signal of neurotrophins. The neurotrophin agonist compounds are suitable for, e.g., preparation of pharmaceutical compositions useful in the treatment of:

i) neurodegenerative, inflammatory, toxic, traumatic, or vascular disorders of the central, peripheral, or autonomic nervous system (such as Alzheimer Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Huntington disease, multiple sclerosis, epilepsy, Down syndrome, nervous deafness, Ménière's disease), neural damages secondary to hypoxia, ischaemia, burns, chemotherapy, toxic compounds of various origin (including alcohol), infections (such as polio or HIV virus), trauma (including surgical trauma) originating axotomy of motoneurons, sensorial, motor, or sensorimotor neuropathies, or autonomic dysfunctions secondary to diverse pathologies (such as diabetes, renal insufficiency, or other systemic diseases), genetic disorders (such as Charcot-Marie-Tooth disease, Refsum disease, abetalipoprotenemia, Tangier disease, Krabbe disease, metachromatic leukodystrophy, Fabry disease, Dejerine-Softas disease), nervous pathologies of diverse origin (such as diffuse athrophy of cerebral cortex, Lewy body dementia, Pick's disease, mesolimbocortical dementia, neuronal ceroid lipofuscinosis, thalamic degeneration, cortico-striatal-spinal degeneration, cortico-basal ganglionic degeneration, cerebro-cerebellar degeneration, familial dementia with spastic paraparesis, pdlyglucosan bodies disease, Shy-Drager synfrome, olivopontocerebellar atrophy, progressive supranuclear palsy, deforming muscular dystony, Hallervorden-Spatz disease, Meige's syndrome, familial shivering, Gilles de la Tourette syndrome, chorea-acanthocytosis syndrome, Friedreich's ataxia, Holmes' corticocerebellar familial atrophy, Gerstmann-Straussler-Scheinker disease, progressive spinal muscular atrophy, spastic paraplegia, peroneal muscular atrophy, hypertrophic interstitial polyneuropathy, polyneuritic ataxic heredopathy), some ocular pathologies (such as optic nerve neuropathies, retinal degeneration, ophtalmoplegy, glaucoma), corneal diseases of diverse origin (such as neurotrophic, ulcers, post-traumatic or post-infective corneal disorders), pathologies from reduced motility of the gastro-intestinal tract or from urinary bladder atony (such as interstitial cystitis or diabetic cystitis), endocrine neoplastic pathologies (such as prolactinoma), clinical conditions in which stimulation of learning processes is advantageous (in particular, in dementias and in post-traumatic conditions), besides all pathological conditions originating from apoptotic processes of neural cells; ii) acquired immunodeficiency diseases due to reduced or absent bioavailability of NGF (such immunodificiency of ageing); iii) conditions in which stimulation of neoangiogenesis may be advantageous (such as myocardial infarction, stroke, cerebral aneurysms, gastro-duodenal ulcers, wound healing, peripheral vasculopathies); iv) certain ocular pathologies (such as corneal pathologies of diverse origin and glaucoma).

The present 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I), and their dimers of general formula (II) and (III) above reported, are also suitable for the preparation of culture and storage media useful for conservation of explanted corneas destined to transplantation.

Moreover, when labelled with suitable reagents (contrast agents, radioisotopes, fluorescent agents, etc.), and possibly processed with any other procedure useful for medical imaging purposes, the present 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I), and their dimers of general formula (II) and (III), may be used for the imaging analysis of tissues and organs containing neurotrophine receptors, either in vitro or in vivo. In particular such labelled compounds may be used either for monitoring the use and efficacy of drugs or for the diagnosis of mammal diseases in which the neurothrophine receptors are involved.

In general, the present compounds having neurotrophin agonistic activity, in particular NGF agonistic activity, were proven adequate to substitute for neurotrophin and NGF biologic activity.

Furthermore, the present neurotrophin agonistic compounds can be used to promote in vivo, in vitro, or ex vivo growth and/or survival of neural cells, including, but not limited to: dopaminergic, cholinergic, sensorial neurons, striatal cells, cortical cells, cells of the corpus striatum, hippocampus, cerebellum, olfactory bulbs, peri-aqueductal cells, cells of the raphe nuclei, of the locus coeruleus, of the dorsal root ganglia, sympathetic neurons, lower motoneurons, nervous stem cells, or cells anyhow deriving from the neural plaque.

The following examples are reported to give a non-limiting illustration of the present invention.

EXAMPLE 1 Preparation of methyl 3-benzyl-2-oxo-(1S,5S,7R)-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-exo-carboxylate (compound of formula (I) where X=O, R₁=H, R₂=Bn, R₆=(R)—COOMe) (Compound 1)

A solution of R,R tartaric anhydride 4 (4 g) (prepared as reported by Lucas H, J., Baumgarten W., J. Am. Chem. Soc., 1941, 63, 1654) in anhydrous dichloromethane (23 ml) and 3a (where X=X=OMe, R₁=H, R₂=H, R₃=Bn,) (3 g) prepared as reported (Kermak, W. O.; Perkin, W. H.; Robinson, R. J. Chem. Soc., Trans, 1922, 121, 1872) were reacted at r.t. for 15 h. After evaporation of the solvent 7a (7 g), is obtained as an oil. To the crude product 7a in CH₃OH (40 ml), thionyl chloride is added dropwise (0.8 ml) at 0° C. and then the mixture heated at 60° C. for 15 h. After evaporation of solvent, the crude product dissolved in toluene (8 ml) is quickly added to a refluxed suspension of (1.6 g) H₂SO₄/SiO₂ (H₂SO₄ 30% by weight) in toluene (12.5 ml). After 15 min, one third of the solvent is distilled off and the remaining hot mixture is filtered on a short pad of NaHCO₃. After evaporation of the solvent, the crude product was purified by chromatography giving the pure compound of the title (2.8 g).

¹H NMR (CDCl₃) δ 7.32-7.16 (m, 5H), 5.84 (d, J=2.0 Hz, 1H), 4.96 (s, 1H), 4.74 (s, 1H), 4.52 (s, 2H), 3.77 (s, 3H), 3.34 (dd, J₁=12.0 Hz, J₂=2.0 Hz, 2H), 3.08 (J=12.0 Hz, 1H). P.f. 82, [α]²⁵ _(D)=−49 (c 1.0, CHCl₃)

EXAMPLE 2 Preparation of methyl (1R,5R,7S)-3-benzyl-2-oxo-6,8-dioxa-3-azabicylo[3.2.1]octane-7-exo-carboxylate (compound of formula (I) where X=O, R₁=R₂=H, R₃=Bn, R₆=(S)—COOMe) (Compound 191)

Following the same procedure of Example 1, starting from anhydride S,S tartaric 4, the compound of the title is obtained.

¹H NMR (CDCl₃) δ 7.40-7.10 (m, 5H), 5.85 (d, J=2.0 Hz, 1H), 4.97 (s, 1H), 4.74 (s, 1H), 4.52 (s, 2H), 3.79 (s, 3H), 3.34 (dd, J₁=12.0 Hz, J₂=2.0 Hz, 2H), 3.09 (J=12.0 Hz, 1H). P.f. 83, [α]²⁵ _(D)=+48 (c 1.0, CHCl₃)

EXAMPLE 3 Preparation of methyl (1S,5S,7R)-3-benzyl-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-exo-carboxylate (compound of formula (I) where X=R₁=R₂=H, R₃=Bn, R₆=(R)—COOMe) (compound 40)

A solution of BH₃.Me₂S (1 M, 2.5 ml,) was slowly added at 0° C. to a solution in anhydrous THF (65 ml) of compound of formula (I) where X=O, R₁=H, R₂=H, R₃=Bn, R₆=(R)—COOMe (compound 1) (2.8 g) prepared as described above in Example 1. The mixture was stirred for 18 h at r.t. and then ethanol (3 ml), NaOH solution (3M, 2 ml) and H₂O (150 ml) were added. After extraction with diethylether, the organic phase was separated and evaporated giving, after chromatography, the pure compound of the title (2 g) as colorless oil.

¹H NMR (CDCl₃) δ 7.30-7.23 (m, 5H), 5.62 (s, 1H), 4.78 (s, 1H), 4.60, (s, 1H), 3.74 (s, 3H), 3.55 (pd, 2H), 2.84 (d, J=13 Hz, 1H), 2.76 (d, J=10 Hz, 1H), 2.50 (dd, J₁=10 Hz, J₂=2 Hz, 1H), 2.30 (d, J=11 Hz, 1H). [α]²⁵ _(D)=−60 (c 1.0, CHCl₃).

EXAMPLE 4 Preparation of methyl (1S,5S,7R)-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-exo-carboxylate (compound of formula (I) where X=R₁=R₂=R₃=H, R₆=(R)—COOMe) (Compound 34)

To a suspension of compound of formula (I) where X=R₁=R₂=H, R₃=Bn, R₆=(R)—COOMe) (compound 40) (2 g) prepared as described above in Example 3 and Pd/C 10% (1.3 g) in methanol (40 ml), is added ammonium formiate (2.4 g). The mixture left at reflux for 1 h, was filtered on Celite and washed with CH₃OH. The solution is evaporated to give the compound of the title (1.3 g), as colorless oil. ¹H NMR (CDCl₃), δ 5.53 (s, 1H), 4.72 (s, 1H), 4.49 (s, 1H), 3.71 (s, 3H), 3.17 (dd, J₁=13.6 Hz, J₂=1.8 Hz, 1H), 2.83 (m, 2H), 2.68 (d, J=13.6 Hz, 1H), 2.55 (br, 1H). [α]²⁵ _(D)=−55 (c 0.7, CHCl₃).

EXAMPLE 5 Preparation of acid (1S,5S,7R)-6,8-dioxa-3-azabicylo[3.2.1]octane-7-exo-carboxylic (compound of formula (I) where X=R₁=R₂=R₃=H, R₆=(R)—COOH) (Compound 32)

The compound of formula (I) where X=R₁=R₂=R₃=H, R₆=(R)—COOMe (Compound 34) prepared as described in Example 4 (0.5 g) was dissolved in a solution of HCl (4N, 12 ml). After 18 h at r.t., the solution was evaporated obtaining the title compound as HCl salt (0.5 g).

[α]²⁵ _(D)=−38.3 (c 1.1, H₂O); ¹H NMR (D₂O) δ 5.95 (s, 1H), 5.06 (s, 1H), 5.04 (s, 1H), 3.58 (m, 2H), 3.34 (m, 2H);

EXAMPLE 6 Preparation of methyl (1S,5S,7R)-3-ter-butoxycarbonyl-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-exo-carboxylate (compound of formula (I) where X=R₁=R₂=H, R₃=Boc, R₆=(R)—COOMe) (Compound 42)

DIPEA (0.8 ml) and (BOC)₂O (1.1 g) were added to a solution in CH₂Cl₂ anhydrous (9 ml) and ethanol (3 ml) of the compound of formula (I) wherein X=R₁=R₂=R₃=H, R₆=(R)—COOMe (Compound 34) (0.8 g) prepared as described in Example 4. The reaction mixture was left for 18 h at r.t., the solvent was evaporated and the residue was treated with a solution of NaHSO₃ (5%) and extracted with diethylether. After evaporation of the solvent, the crude product was purified by chromatography to give the title compound (0.8 g) as white solid.

¹H NMR (CDCl₃) δ 5.64 and 5.58 (rotamers) (s, 1H), 4.65 and 4.60 (rotamers) (s, 1H), 4.51 (s, 1H), 3.72 (s, 3H), 4.00-3.60 (m, 2H), 3.20 (m, 1H), 2.92 (m, 1H), 1.43 (s, 9H).

EXAMPLE 7 Preparation of (1S,5S,7R)-3-ter-butoxycarbonyl-6,8-dioxa-7-exo-hydroxymethyl-3-azabicyclo[3.2.1]octane (compound of formula (I) where X=R₁=R₂=H, R₃=Boc, R₆=(R)—CH₂OH) (Compound 62)

To a solution in MeOH (15 ml) of the compound of formula (I) where X=R₁=R₂=H, R₃=Boc, R₆=(R)—COOMe) (Compound 42) (0.8 g) prepared as described in Example 6, at 0° C., NaBH₄ (0.6 g) was added in small portions. After 10 min at r.t., the mixture was evaporated, and the crude product was purified by chromatography to give the compound of the title (0.5 g) as a colourless oil. [α]²⁵ _(D)−30 (c 1.0, MeOH).

¹H NMR (CDCl₃) δ 5.50 and 5.44 (rotamers) (s, 1H), 4.32 and 4.27 (rotamers) (s, 1H), 4.18 (m, 1H), 3.88-3.67 (m, 2H), 3.56 (d, J=5.5 Hz, 2H), 3.21 (m, 1H), 2.96 (m, H), 1.92 (b, 1H), 1.43 (s, 9H).

EXAMPLE 8 Preparation of (1S,5S,7R)-3-(9-Fluorenylmethoxycarbonyl)-7-endo-hydroxymethyl-6,8-dioxa-3-aza-bicyclo[3.2.1]octane (compound of formula (I) where X=R₁=R₂=H, R₃=Fmoc, R₆=(R)—CH₂OH) (Compound 61)

To a solution of 2,3-O-isopropylidene-D-erithrose (R,R) 5 (1.8 g) in THF (prepared from D-Arabinose, as reported by Thompson, D. K.; Hubert, C. N.; Wightman, R. H. Tetrahedron 1993, 49, 3827-3840) 2,2-diethoxyethylamine 3a (where W=W=OEt, R₁=R₂=R₃=H) (1.7 ml) a 0° C., NaBH(OAc)₃ (3.1 g) was added in small portions. After 18 h a r.t., the mixture is diluted with a saturated solution of NaHCO₃ and extracted with ethyl acetate. The organic phase was evaporated giving an oil, which was chromatographed to give the product 8a (where W=W=OEt, R₁=R₂=R₃=H) as yellowish oil (1.9 g).

[α]²⁰ _(D) −8.4 (c 0.54, CHCl₃); ¹H NMR (CDCl₃) δ 4.83 (br, 2H), 4.59 (t, J=5.5 Hz, 1H), 4.32 (m, 2H), 3.75-3.45 (m, 6H), 3.05-2.83 (m, 2H), 2.79 (d, J=5.5 Hz, 2H), 1.44 (s, 3H), 1.34 (s, 3H), 1.21 (t, J=7.0 Hz, 6H).

To a solution of 8a (where W=W=OEt, R₁=R₂=R₃=H) (1.7 g) in acetone (40 ml) Fmoc-O-Su (2.1 g) and an aqueous solution of Na₂CO₃.H₂O (0.75 g in 40 ml) were added at 0° C. The mixture was left at r.t. for 18 h, and extracted with CH₂Cl₂, then the solvent was evaporated and the residue was chromatographed to give the product 8a (where W=W=OEt, R₁=R₂=H, R₃=Fmoc) as yellowish oil (2.2-9). [α]²⁰ _(D) −34 (c 0.38, MeOH); ¹H NMR (CDCl₃) δ7.73 (d, J=7.3 Hz, 2H), 7.56 (m, 2H), 7.34 (m, 4H), 4.63 (m, 2H), 4.47-4.14 (m, 3H), 4.19 (t, J=4.9 Hz, 1H), 3.74-3.02 (m, 10H), 1.42-1.04 (m, 12H);

Compound 8a (where W=W=OEt, R₁=R₂=H, R₃=Fmoc) (1.9 g) dissolved in trifluoroacetic acid (8 ml) was left aside for 0.18 h a r.t. After evaporation of TFA, the crude compound, dissolved in MeOH, was filtered on as short pad of NaHCO₃, then the solvent was evaporated and the residue was chromatographed to, give the title product as a white solid (1 g).

M.p. 41-42° C.; [α]²⁰ _(D) −32 (c 0.5, CHCl₃); ¹H NMR (CDCl₃) δ7.77 (d, J=7.0 Hz, 2H), 7.57 (d, J=7.0 Hz, 2H), 7.38 (m, 4H), 5.51 (s, 1H), 4.92-2.95 (m, 12H).

EXAMPLE 9 Preparation of acid (1S,5S,7S)-3-(9-Fluorenylmethoxycarbonyl)-6,8-dioxa-3-aza-bicyclo[3.2.1]octan-7-endo carboxylic (compound of formula (I) where X=R₁=R₂=H, R₃=Fmoc, R₆=(S)—COOH) (Compound 39)

To a solution of the compound of formula (I) where X=R, =R₂=H, R₃=Fmoc, R₆=(R)—CH₂OH (compound 61) (0.9 g) prepared according to the Example 8, in acetone (75 ml) was added the Jones reagent at 0° C., [prepared by slow addition of H₂SO₄ (2.8 ml) to a solution of CrO₃ (1.5 g) in H₂O (20 ml) a 0° ]. The mixture was left for 18 h at r.t and then was added with isopropanol, filtered on Celite and evaporated. The crude product dissolved in EtOAc (45 ml) was extracted with 10% NaHCO₃ in water. After separation, the aqueous phase was acidified at pH 1 with HCl and extracted with EtOAc. Evaporation of the organic phase gave a crude product which was chromatographed to give the compound of the title (0.7 g) as a white solid.

M.p. 79-82° C.; [α]²⁰ _(D) −53 (c 0.5, CHCl₃); ¹H NMR (CDCl₃) δ 7.75 (m, 2H); 7.53 (d, J=7.0 Hz, 2H); 7.38 (m, 4H); 5.56 (s, 1H); 4.74-4.45 (m, 4H); 4.23-3.91 (m, 4H); 3.29-3.11 (m, 2H).

EXAMPLE 10 Preparation of (1R,5R,7R)-3-(9-Fluorenylmethoxycarbonyl)-6,8-dioxa-3-aza-bicyclo[3.2.1]octan-7-endo carboxylic acid (compound of formula (I) where X=R₁=R₂=H, R₃=Fmoc, R₆=(R)—COOH) (compound 218)

A solution of (1R,5R,7S)-3-(9-fluorenylmethoxycarbonyl)-7-endo-hydroxymethyl-6,8-dioxa-3-aza-bicyclo[3.2.1]octane (compound of formula (I) where X=R₁=R₂=H, R₃=Fmoc, R₆=(S)—CH₂OH) (1.8 g), prepared from (S,S) erythrose 5 (obtained starting from L-arabinose) with the same procedure above described in the Example 8 for its enantiomer, was treated as above described in the Example 9 for its enantiomer, to give 1.4 g of the title compound as white solid.

M.p. 71-81° C.; [α]²⁰ _(D) +52.9 (c 0.50, CHCl₃).

EXAMPLE 11 Preparation of methyl 3-benzyl-5-phenyl-2-oxo-(1S,5S,7R-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-exo-carboxylate (compound of formula (I) where X=O, R₁=Ph, R₂=H, R₃=Bn, R₆=(R)—COOMe) (Compound 27)

To a solution of 3b (2.4 g) (where X=O, R₁=Ph, R₂=H, R₃=Bn,) (prepared according the procedure reported by R Simonoff and W. H Hartung, J. Am. Pharm. Assoc., 35, 306, 1946) in dry CH₂Cl₂ (20 ml), (R,R) 6 acid tartaric derivative (2.49 g, 5.33 mmol) and DIPEA (5.4 ml) were added. The mixture was stirred at r.t. for 2 h, the solvent was evaporated to give an oil which was extracted in ethyl acetate. The solution was washed with solution of 5% KHSO₄, and 5% NaHCO₃ in water. After evaporation of the solvent the residue was purified by chromatography to give 8b (where X=O, R₁=Ph, R₂=H, R₃=Bn,) (3.2 g) as colourless oil.

¹H NMR δ7.90-7.85 (m, 2H), 7.61-7.22 (m, 8H), 5.39 (d, J=5.1 Hz, 1H), 5.11 (d, J=5.1 Hz, 1H), 4.88-4.10 (m, 4H), 3.80 (s, 3H), 1.49 (s, 3H), 1.31 (s, 3H).

A solution of 8b (3.2 g) (where X=O, R₁=Ph, R₂=H, R₃=Bn,) in toluene (80 ml) was quickly added to a suspension of H₂SO₄/SiO₂ (30% w/w, 1.4 g) in toluene at reflux (120 ml). After 15 min one third of the solvent was distilled off and the hot remaining mixture was filtered on a short pad of NaHCO₃. After evaporation of the solvent the residue was purified by chromatography to give 2.4 g of the title compound as colorless solid.

M.p. 113-114° C. [α]²⁵ _(D) −64.0 (c 1, CDCl₃). ¹H NMR δ 7.62-7.59 (m, 2H), 7.41-7.24 (m, 8H), 5.16 (s, 1H), 4.92 (s, 1H), 4.61 (m, 2H), 3.74 (s, 3H), 3.46 (m, 2H).

EXAMPLE 12 Preparation of methyl 3-benzyl-5-phenyl-(1S,5S,7R)-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-exo-carboxylate (compound of formula (I) where X=R₂=H, R₁=Ph, R₃=Bn, R₆=(R)—COOMe) (Compound 120)

To a solution in dry THF (25 ml) of the compound of formula (I) where X=O, R₁=Ph, R₂=H, R₃=Bn, R₆=(R)—COOMe) (compound 27) prepared as described in Example 11 (2.5 mmol), at 0° C., BH₃.Me₂S (10 M 0.5 ml, 4.9 mmol) was added dropwise. The mixture was left aside for 16 hr and then EtOH (1 ml), 3 M NaOH 1 ml) and H₂O (20 ml) were added. After extraction with diethylether, and evaporation of the solvent the residue was purified by chromatography to give 1 g of the compound of the title as colorless solid.

M.p. 97° C. [α]_(D) ²⁵=13.0 (c 1, CHCl₃). ¹H NMR δ 7.72-7.58 (m, 2H), 7.52-7.19 (m, 8H), 5.00 (s, 1H), 4.86 (s, 1H), 3.75 (m 2H), 3.78 (s, 3H), 3.62 (m, 2H), 3.16 (d, J=11.2, 4H), 2.93 (d, J=11.6, 2H), 2.63 (d, J=11.0, 2H).

EXAMPLE 13 Preparation of methyl (1S,4S,7R)-3,4-Dibenzyl-2-oxo-6,8-dioxa-3-azabicyclo[3,2,1]octane-7-exo-carboxylate (compound of formula (I) where X=O, R₁=H, R₂=(S)Bn, R₃=Bn, R₆=(R)—COOMe) (Compound 12)

To a solution of L-phenylalaninol 3c (where W=H, W=OH, R₁=H, R₂=Bn, R₃=H) (5 g) in MeOH (150 ml) benzaldehyde (3.3 ml) were added. The reaction mixture was stirred at r.t. for 1 h, then 1.2 g of NaBH₄, were added in small portions in 2 hr at 0° C. The solvent was evaporated and the residue extracted with 50 ml of HCl at pH=2. The aqueous solution was extracted with Et₂O, treated with Na₂CO₃ until pH=9 and then extracted with CHCl₃. The organic phase evaporated gave N-benzyl-(L)-phenylalaninol as white solid (7 g) 3c (where W=H, W=OH, R₁=H, R₂=Bn, R₃=Bn)

¹H NMR (CDCl₃) δ, ppm: 7.34-7.06 (m, 10H), 3.73 (s, 2H), 3.31 (dd, J=6.2, 12.5 Hz, 1H), 3.00-2.81 (m, 1H), 2.80-2.66 (m, 2H). 2.62 (dd, J=6.2, 12.5 Hz, 1H)

To a solution of N-benzyl-(L)-phenylalaninol 3c (2.8 g) in 23 ml of CHCl₃ at 0° C. DIPEA (4 ml), HOBt (2.1 ml) and a solution of methyl ester of (2R,3R)-2,3-O-isopropylidentartaric acid (6) (2.4 g) in 23 ml of CHCl₃, were added. Then 1.7 g of DIPC were added. After 72 hr at r.t, the solvent was evaporated and the crude product residue was purified by chromatography to give a yellowish solid (2.4 g) 9c (where W=H, W=OH, R₁=H, R₂=Bn, R₃=Bn).

[α]_(D) ²⁵−72 (c=0.5, CHCl₃). ¹H NMR (CDCl₃), δ, ppm: (mixture of rotamers 2:1) major δ 7.40-7.05 (m, 10H), 5.28 (d, J=6.0 Hz, 1H), 4.81 (d, J=6.0 Hz, 1H), 4.75 (d, J=16.4 Hz, 1H), 4.0 (d, J=16.4 Hz, 1H), 3.79 (s, 3H), 3.70 (m, 1H), 3.60 (m, 1H), 3.46 (m, 1H), 3.04 (m, 1H), 1.52 (s, 3H), 1.49 (s, 3H).

The compound 9c (where W=H, W=OH, R₁=H, R₂=Bn, R₃=Bn) was oxidized to 10 (where W=O, W=O, R₁=H, R₂=Bn, R₃=Bn) by Swem oxidation. 4.5 g of alcohol (9c) in 20 ml of CH₂Cl₂ were oxidized as usual by treatment with oxalyl chloride, DMSO and DIPEA. After usual work-up compound (10) (5 g) was obtained as yellow solid.

¹H NMR (CDCl₃) δ ppm: 9.44 (s, 1H), 7.40-7.00 (m, 10H), 5.33 (d, J=6.2 Hz, 1H), 4.92 (d, J=6.2 Hz, 1H), 4.89 (d, J=18.7 Hz, 1H), 3.79 (s, 3H), 3.53 (dd, J=9.8, 4.3 Hz, 1H), 3.44 (d, J=18.7 Hz, 1H), 3.41 (dd, J=13.9, 4.3 Hz, 1H), 3.12 (dd, J=13.9, 9.8 Hz, 1H), 1.54 (s, 3H), 1.45 (s, 3H).

The product was added in toluene (15 ml), to a suspension of 2.5 g SiO₂ and H₂SO₄ in 30 ml of refluxing toluene; After 30 min, After 15 min one third of the solvent was distilled off and the hot remaining mixture was filtered on a short pad of NaHCO₃. After evaporation of the solvent the residue was purified by chromatography to give 3.2 g of the title compound.

¹H NMR (CDCl₃) δ ppm: 7.40-7.15 (m, 8H), 7.03 (m, 2H), 5.51 (s 1H), 5.33 (d, J=15.0 Hz, 1H), 4.97 (s, 1H), 4.71 (s, 1H), 4.03 (d, J=15.0 Hz, 1H), 3.75 (s, 3H), 3.32 (dd, J=10.7, 3.7 Hz, 3H), 3.15 (dd, J=13.5, 3.7 Hz, 1H), 2.75 (dd, J=13.5, 10.7 Hz, 1H)

EXAMPLE 14 Preparation of (1S,4S,7R)-3,4-Dibenzyl-6,8-dioxa-7-exo-hydroxymethyl 3-azabicyclo[3,2,1]octane (compound of formula (I) where X=R₁=H, R₂, (S)Bn, R₃=Bn, R₆=(R)—CH₂OH) (Compound 184)

To a solution in 100 ml of anhydrous THF of the compound of formula (I) where X=O, R₁=H, R₂=(S)Bn, R₃=Bn, R₆=(R)—COOMe (compound 12) (4 g), prepared as described in Example 13, a solution BH₃.SMe₂ (3 ml, 10 M) in THF was added. After 38 hr at r.t. the reaction mixture was treated with dry EtOH (6 ml) and 10% of NaOH (6 ml), then diluted with 50 ml of water and extracted with Et₂O. After evaporation of the solvent the residue was purified by chromatography to give 1.7 g of the title compound as yellowish solid. [α]_(D) ²⁵−59 (c=0.2, CHCl₃)

¹H NMR (CDCl₃) δ, ppm: 7.40-7.00 (m, 10H), 5.11 (s, 1H), 4.39 (t, J=5.1 Hz, 1H), 4.24 (s, 1H), 3.81 (d, J=13.6 Hz, 1H), 3.63 (d, J=13.6 Hz, 1H) 3.52 (m, 2H), 3.00 (m, 1H) 3.00-2.80 (m, 2H), 2.94 (d, J=11.6 Hz, 1H), 2.45 (dd, J=11.6, 1.8 Hz, 1H)

EXAMPLE 15 Preparation of Dimer of Formula (II) where R₁=R₁′=H, R₂=R₃=R₂′=Bn, R₆=(R)—COOMe (Compound 348)

0.1 ml of DIPEA were added to a solution in 0.3 ml of CH₂Cl₂ of the compound of formula (I) where X=R₁=H, R₂=(S)Bn, R₃=Bn, R₆=(R)—COOH (Compound 188) (0.1 g) obtained by hydrolysis of the corresponding methyl ester (Compound 172) according to the procedure in Example 5. Then, 0.2 g of PyBroP at 0° C. and 0.05 g (0.209 mmol) of the compound of formula (I) where X=R₁=R₃H, R₂=(S)—Bn, R₆=(R)—COOMe (Compound 178) were added. The mixture was stirred overnight, the solvent evaporated and the residue dissolved in 50 ml of AcOEt. After evaporation of the solvent the residue was purified by chromatography to give 0.07 g of the title compound as white solid.

EXAMPLE 16 Preparation of Dimer of Formula (III) where X=O, R₁=R₁′=p-NO₂Ph, R₂=R₂′=H, R₃=R′₃=Ph, Q′=(CONH)CH₂)₆CONH) (Compound 441)

20 mg of (1R,5S,7R)-5-(4-Nitro-phenyl)-3-phenyl-6,8-dioxa-3-aza-bicyclo[3.2.1]octane-7-carboxylic acid methyl ester of formula (I) (Compound 31) (0.054 mmol) were added to 125.5 mg (1.08 mmol, 20 eq) of 1,6-diamino-hexane and the mixture heated at 65° C. overnight. The crude is purified by chromatography (CH₂Cl₂-MeOH, 20:1+NEt₃ 1%), thus obtaining 8 mg (0.018 mmol, 34%) of a yellow solid corresponding to (1R,5S,7R)-5-(4-nitro-phenyl)-3-phenyl-6,8-dioxa-3-aza-bicyclo[3.2.1]octane-7-(6-amino-hexyl)amide, i.e. the compound of formula (I) where X=O, R₁ p-NO₂Ph, R₂=H, R₃=Ph, R₆=CONH(CH₂)₆NH₂ (Compound 189) (R_(f)=0.32) and 4 mg (0.0051 mmol, 10%) of an orange solid corresponding to the dimeric compound of formula (III) of the title (Rf=0.67).

Compound 189: ¹H NMR (CDCl₃, δ): 832 (d, 2H, J=8.4 Hz), 7.83 (d, 2H J=8.8 Hz), 7.30-7.22 (m, 2H), 6.90-6.79 (m, 3H), 6.25 (m, 1H), 5.05 (s, 1H), 4.74 (s, 1H), 3.81-3.70 (m, 2H), 3.28 (d, 1H, J=9.8 Hz,), 3.20-3.10 (m, 2H), 2.92 (d, 1H, J=11.6 Hz), 2.61 (m, 2H), 1.78-1.15 (m, 10H).

dimeric compound of formula (III) of the title: ¹H NMR (CDCl₃, δ): 88 (d, 4H, J=8.8 Hz), 7.82 (d, 4H, J=10 Hz), 7.31-7.24 (m, 4H), 6.91-6.80 (m, 6H), 6.25 (m, 2H), 5.05 (s, 2H), 4.75 (s, 2H), 3.81-3.71 (m, 4H), 3.29 (d, 2H, J=11.6 Hz,), 3.20-3.10 (m, 4H), 2.92 (d, 2H, J=11.6 Hz), 1.54 (m, 4H), 1.23 (m, 4H).

Biological Activity

The biological activity of 3-aza-bicyclo(3.2.1)octanes of formula (I) and their dimeric forms of formula (II) and (III) was evaluated in different assays: induction of survival of PC12 cells in serum-free conditions, induction of proliferative activity in PC3 prostatic carcinoma cell line, induction of VGF polypeptide synthesis, displacement of 125I-NGF binding to specific surface receptor, and induction of Trk-A autophosphorylation. In all of these assays human recombinant (hr)NGF was used as internal standard.

Effect of Compounds on PC12 Cell Survival in Serum-Free Conditions.

The biological activity of 3-aza-bicyclo(3.2.1)octanes of formula (I) and their dimeric forms of formula (II) and (III) was tested as ability to induce the survival of PC12 cells in serum-free conditions by using hrNGF as internal standard. PC12 cells were detached from tissue flasks with PBS-EDTA (physiological saline solution added with ethylendiaminotetraacetic acid) and washed once with PBS to avoid residual amounts of serum. The cells were then diluted in RPMI-1640 medium without phenol red supplemented with penicillin and streptomycin and cultured in 96 well plates at the final concentration of 5×10³/well. Standard curve was performed by adding in triplicate cultures different concentrations of hrNGF, in the range between 1-25 ng/ml. The compounds were instead added, in triplicate, at the final concentrations of 1, 10, 100 μM. The cells were then cultured for 60 hours at 37° C. in a humidified, 5% CO₂, atmosphere. Then 10 μl of (3-[4.5-dimethylthiazol-2yl]-2.5-diphenyltetrazolium bromide (MTT, 0.5 mg/ml in isopropanol) were added to each well and plates, protected from the light, were left at 37° C. for 4 hours. At the end of incubation, 100 μl of 50% dimethylformamide (in 20% SDS, pH 7.4) were added to each well. Colorimetric reaction was detected with a 96 well plate reader by recording the absorbance at 570 nm. Results were expressed as survival induced by compounds/spontaneous survival*100

FIG. 1 shows the results obtained with 10 μM of the most representative compounds and with 1 nM of hrNGF.

Effect of Compounds on Proliferative Activity of PC3 Cell Line.

The ability of 3-aza-bicyclo(3.2.1)octanes of formula (I) and their dimeric forms of formula (II) and (III) with substitutions reported in Table 1-4 to induce proliferation of PC3 cell line, in serum-free conditions, was tested by using hrNGF as internal standard.

PC3 cells were cultured in triplicate in 24 well plates at the final concentration of 10⁴ cells/ml (final volume of 500 μl) in RPMI 1640 medium in the presence or absence of 1, 10, 100 μM of the compounds or of different concentration (between 1-25 ng/ml) of hrNGF as internal standard. Cells were incubated for 60 hours in humidified, 5% CO₂, atmosphere. At the end of incubation 0.5 μCi of ³H-thymidine were added to each well for 8 hours. Cells were then washed 6 times with PBS, lysed with 0.1% Triton-X100 in 0.1 M phosphate buffer, and the radioactivity was recorded in a β-scintillation counter. Results were expressed as ratio between ³H-thymidine incorporation (mean±SD) of stimulated cultures and ³H-thymidine incorporation of non stimulated cultures. FIG. 2 shows the results obtained with 10 μM of selected compounds or with 1 nM hrNGF as internal standard.

Induction of VGF Production by PC12 Cells

The ability of 3-aza-bicyclo(3.2.1)octanes of formula (I) and their dimeric forms of formula (II) and (III) with substitution reported in Table 1-4 was tested also as ability to induce VGF production by PC12 cells. 5×10⁶ PC12 cells were cultured in the presence or absence of 1, 10, 100 μM of the compounds or of 4 nM hrNGF as internal standard for 24 hours in humidified, 5% CO₂, atmosphere. Cells were lysed in 0.25% NP-40 in PBS supplemented with 1 mM PMSF (phenyl-methyl) and 1 mM leupeptin and protein concentration was measured in each sample by Bradford assay. Equal amounts of proteins (30 μg) were loaded in 8% SDS-polyacrilamide gel, electrophoresed, blotted onto nitrocellulose membrane and stained with monoclonal antibodies anti-VGF followed by peroxidase-conjugated anti-mouse IgG. Reaction was visualized by Enhanced Chemiluminiscent Reagent (ECL, Amersham) following the manufacturer instruction.

FIG. 3 shows the results obtained with 10 μM of the selected (n. 91, 9, 323, 270) compounds or with 10 nM hrNGF. VGF is induced by the selected compounds as well as by hrNGF.

Displacement of ¹²⁵I-NGF Binding to PC12 Cells

The ability of selected compounds to displace the binding of NGF to specific surface receptor was evaluated through the classic binding techniques of iodinated ligand.

PC12 cells were detached from tissue flasks with PBS-EDTA, washed with HKR medium (10 mM Hepes, 125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 1 g/l glucose, 1 g/l BSA) and incubated in triplicate in HKR medium with 0.1 nM ¹²⁵I-NGF in the presence or absence of variable concentrations of the compounds to be assayed or of hrNGF as internal standard. Displacement curve was obtained by analyzing the resultant cell bound radioactivity in the presence of the compounds or of hrNGF with adequate software (Graphit 4).

FIG. 4 a shows the displacement curve obtained with the compound n.9 used as competitor The analysis of data revealed a Kd of 165 nM±0.05. FIG. 4 b shows the displacement curve obtained by using hrNGF as competitor. The analysis of data revealed a Kd of 114 pM±0.01 as already reported.

Trk-A Autophosphorylation

To evaluate the ability of the compounds 3-aza-bicyclo(3.2.1)octanes of formula (I) and their dimeric forms of formula (II) and (III) reported in Table 14 to induce Trk-A autophosphorylation, PC12 cells were cultured in medium supplemented with 5% FBS for 48 hours, washed and equilibrated in serum-free medium for 2 hours. 2.5×10⁶ cells were then stimulated with 10 μM of selected compounds for 30 min or with 10 nM hrNGF as positive control. Cells were then lysed with 0.5% Triton-X100 in PBS supplemented with protease inhibitors (PMSF, aprotinin, pepstatin, leupeptin) and phosphatase inhibitors. Protein concentrations in each sample was evaluated by Bradford assay and equal amounts (50 μg) of proteins were loaded onto SDS-polyacrilamide gel, electrophoresed and blotted onto nitrocellulose membrane. Membranes was stained with rabbit anti-(Tyr 490 and Tyr 674/675) phosphorylated Trk-A (Cell Signaling Technology) used at the final dilution of 1:1000. After washing, membranes were stained with HRP-conjugated anti-rabbit IgG and the reaction was visualised by using ECL reagents following manufacturing instructions.

FIG. 5 shows the results obtained with the compounds 272, 325, 9, 91 and with hrNGF used as internal standard. The selected compounds are able to induce Trk-A autophosphorylation thus triggering the transduction of biological signals.

Synergic Activity

The synergic activity of multiple combinations of 3-aza-bicyclo(3.2.1)octanes of formula (I) and their dimeric forms of formula (II) and (III) was evaluated in the PC12 survival assay in serum-free condition.

PC12 cells were seeded in 96 well plates at the concentration of 5×10³/well and cultured in triplicate in the presence or absence of 5 μM of selected compounds or of multiple combination of the same compounds at the final concentration of 10 μM. 0.5 nM hrNGF was used as internal standard. After 60 hours at 37° C. in a humidified, 5% CO₂, atmosphere, 10 μl of (3-[4.5-dimethylthiazol-2yl]-2.5-diphenyltetrazolium bromide (MTT, 0.5 mg/ml in isopropanol) were added to each well and plates, protected from the light, were left at 37° C. for 4 hours. At the end of incubation, 100 μl of 50% dimethylformammide (in 20% SDS, pH 7.4) were added to each well. Colorimetric reaction was detected with a 96 well plate reader by recording the absorbance at 570 nm. Results were expressed as survival induced by compounds/spontaneous survival*100. FIG. 6 shows as selected combinations of 2 compounds (91 and 325) induce survival activity higher than the addition of activities induced by the single compound. 

1. A pharmaceutical composition comprising as active principle at least one among the 3-aza-bicyclo[3.2.1]octane derivatives of general formula (I), or mixtures thereof

wherein: R₁ is H, R₂ is selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, arylC₁₋₈alkyl, heterocycleC₁₋₈alkyl, aminoC₁₋₈alkyl, aminoaryl, C₁₋₈alkyloxyaryl, hydroxyaryl, hydroxyC₁₋₈alkyl, carboxyC₁₋₈alkyl, methyloxycarbonylC₁₋₈alkyl, carboxyaryl, carboalkyloxyaryl, alkylcarbamoylaryl and -(side chains of amino acids), or R₃ is selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, arylC₁₋₈alkyl, heterocycleC₁₋₈alkyl, RR′NC₁₋₈alkyl, RR′Naryl, RO—C₁₋₈alkyl, RO(O)C—C₁₋₈alkyl, R(O)C—C₁₋₈alkyl, RC(O)O—C₁₋₈alkyl, RC(O)N(R)C₁₋₈alkyl, RO-aryl, RO(O)C-aryl, R(O)C-aryl RC(O)O-aryl, RC(O)N(R)aryl, —CH(amino acid side-chain)CO₂R, —CH(amino acid side-chain)C(O)NR, —CH(CO₂R)— amino acid side-chain, CH(CONRR′)— amino acid side-chain, Fmoc, Boc and Cbz, R₄, and R₅, equal or different amongst each other, are selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkinyl, cycloalkyl, aryl, heterocycle, arylC₁₋₈alkyl and heterocycleC₁₋₈alkyl, R₆ is selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, arylC₁₋₈alkyl, heterocycle, heterocycleC₁₋₈alkyl; —C(O)R, —C(O)OR, —C(O)NRR′, CH₂OR, CH₂NRR′, —C(O)NH—CH(amino acid side-chain)C(O)OR, CH₂NR-Fmoc, CH₂NR-Boc and CH₂NR—CBz, R and R′, equal or different between each other, are selected from the group consisting of H, C₁₋₈alkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, cycloalkyl, aryl, heterocycle, arylC₁₋₈alkyl; heterocycleC₁₋₈alkyl; protecting group, —C(O)CH-(amino acid side-chain)-NHT, —NH—CH(amino acid side-chain)COOT and —CH(amino acid side-chain)COOT, where T is selected from between H and C₁₋₈alkyl; X is O, α is a double bond, Y and Z, equal or different from each other, are selected from the group consisting of O, S, SO, SO₂ and N—R, wherein R is as above defined; Q is selected from the group consisting of C═O, CH₂, CO—NH—CH (amino acid side-chain)-CO, CONR(CH₂)_(n)CO, CONR—C₂₋₈alkenyl-CO C(O)O(CH₂)CO, CH₂OC(O)(CH₂)CO, and CH₂NRC(O)(CH₂)_(n)CO, wherein n is comprised between 2 and 6, and R is as above defined, Q′ is selected from the group consisting of C(O)OCH₂, C(O)NRCH₂, CH₂OC(O), CH₂NRC(O), CONR(CH₂)_(n)NRCO, CONR—C₂₋₈alkenyl-NRCO, C(O)O(CH₂)_(n)NRCO, CONR(CH₂)_(n)OC(O), CH₂OC(O)(CH₂)_(n)OC(O)CH₂, CH₂NRC(O)(CH₂)_(n)NRC(O)CH₂, CH₂OC(O)(CH₂)_(n)NRC(O)CH₂, CH₂NRC(O)(CH₂)_(n)OC(O)CH₂, CH₂NR(CH₂)_(n)NRCH₂, CH₂O(CH₂)_(n)OCH₂, CH₂O(CH₂)_(n)NRCH₂, and CH₂NR(CH₂)_(n)OCH₂, wherein n is comprised between 2 and 6, and R is as above defined, and where the groups alkyl, alkenyl, alkynyl, cycloalkyl, aryl and the heterocyclic groups above reported, are possibly substituted; and a pharmaceutically acceptable excipient or diluent.
 2. The pharmaceutical composition according to claim 1, wherein Z is O.
 3. The pharmaceutical composition according to claim 1, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclic groups may be substituted with one or more moieties chosen from the group consisting of halogen, cyano, nitro, amino, hydroxy, carboxylic acid, carbonyl and C₁₋₆alkyl.
 4. The pharmaceutical composition according to claim 1, wherein the 3-aza-bicyclo[3.2.1]octane derivatives of formula (I) are selected from the compounds having the following formulas: (I)

Compound X R₁ R₂ R₃ R₆ 1 O H H PhCH₂ (R) —CO₂Me 2 O H H PhCH₂ (S) —CO₂Me 3 O H H PhCH₂

4 O H H PhCH₂

5 O H (S) —Me PhCH₂ (R) —CO₂Me 6 O H (S) —Me PhCH₂ (S) —CO₂Me 7 O H (R) —Me PhCH₂ (R) —CO₂Me 8 O H (R) —Me PhCH₂ (S) —CO₂Me 9 O H (R) —CH₂Ph PhCH₂ (S) —CO₂Me 10 O H (R) —CH₂Ph PhCH₂ (R) —CO₂Me 11 O H (S) —CH₂Ph PhCH₂ (S) —CO₂Me 12 O H (S) —CH₂Ph PhCH₂ (R) —CO₂Me 13 O H (S) —CH₂OBn PhCH₂ (R) —CO₂Me 14 O H (S) —CH₂OBn PhCH₂ (S) —CO₂Me 15 O H (R) —CH₂OBn PhCH₂ (R) —CO₂Me 16 O H (R) —CH₂OBn PhCH₂ (S) —CO₂Me 17 O H (S) —CH₂OH PhCH₂ (R) —CO₂Me 18 O H (S) —CH₂OH PhCH₂ (S) —CO₂Me 19 O H (R) —CH₂OH PhCH₂ (R) —CO₂Me 20 O H (R) —CH₂OH PhCH₂ (S) —CO₂Me 21 O H ═CH₂ PhCH₂ (R) —CO₂Me 22 O H ═CH₂ PhCH₂ (S) —CO₂Me 23 O H (R) —CH₂OH PhCH₂ (S) —CO₂Me

(I)

Com- pound X R₁ R₂ R₃ R₆ 190 O H H PhCH₂ (R) —CO₂Me 191 O H H PhCH₂ (S) —CO₂Me 192 O H (S) —Me PhCH₂ (R) —CO₂Me 193 O H (S) —Me PhCH₂ (S) —CO₂Me 194 O H (R) —Me PhCH₂ (R) —CO₂Me 195 O H (R) —Me PhCH₂ (S) —CO₂Me 196 O H (S) —PhCH₂ PhCH₂ (R) —CO₂Me 197 O H (S) —PhCH₂ PhCH₂ (S) —CO₂Me 198 O H (R) —PhCH₂ PhCH₂ (R) —CO₂Me 199 O H (R) —PhCH₂ PhCH₂ (S) —CO₂Me 200 O H (S) —CH₂CH(Me)₂ PhCH₂ (R) —CO₂Me 201 O H (S) —CH₂CH(Me)₂ PhCH₂ (S) —CO₂Me 202 O H (R) —CH₂CH(Me)₂ PhCH₂ (R) —CO₂Me 203 O H (R) —CH₂CH(Me)₂ PhCH₂ (S) —CO₂Me 204 O H H PhCH₂ (R) —CONHMe 205 O H H PhCH₂ (S) —CONHMe 206 O H (S) —Me PhCH₂ (R) —CONHMe 207 O H (S) —Me PhCH₂ (S) —CONHMe 208 O H (R) —Me PhCH₂ (R) —CONHMe 209 O H (R) —Me PhCH₂ (S) —CONHMe 210 O H (S) —PhCH₂ PhCH₂ (R) —CONHMe 211 O H (S) —PhCH₂ PhCH₂ (S) —CONHMe 212 O H (R) —PhCH₂ PhCH₂ (R) —CONHMe 213 O H (R) —PhCH₂ PhCH₂ (S) —CONHMe 214 O H (S) —CH₂CH(Me)₂ PhCH₂ (R) —CONHMe 215 O H (S) —CH₂CH(Me)₂ PhCH₂ (S) —CONHMe 216 O H (R) —CH₂CH(Me)₂ PhCH₂ (R) —CONHMe 217 O H (R) —CH₂CH(Me)₂ PhCH₂ (S) —CONHMe.


5. The 3-aza-bicyclo[3.2.1]octane derivatives of formula (I) selected from the compounds indicated by the following numbers: 3, 4, 22-23, and 200-217, as defined in claim
 4. 